STUDIES ON PHYSIOLOGICAL FUNCTION OF PLATELET-ACTIVATING FACTOR

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Following results were obtained on the physiological role and cellular signaling of platelet-activating factor (PAF).(1)Cellular signaling via PAF receptorBy using transfected receptor in CHO cells, cellular signaling after PAF application was determined. PAF stimulates phosphoinositide turnover, mitogen-activated protein (MAP) kinase, phospholipase A2, and inhibits adenylate cyclase. From the results of PTX-treatment, PAF receptor appears to couple with two types of G-proteins. Ca-independent activation of MAP kinase was inhibited by wortmannin, suggesting a wortmannin-sensitive molecule such as phosphatidylinositol 3-kinase is present between Gi and MAP kinase.To determine the molecular mechanisms of signal desensitization by PAF,we prepared CHO cells carrying wild-type, C-terminal deletion mutant, and substitution mutant (Ser, Thr to Ala) receptors. We found that C-terminal cytoplasmic tail is not required for forward signal transduction, but it is essential for signal-shut down. A family of GRK (G-protein receptor kinase) may be involved in the phosphorylation of Ser/Thr residues leading to signal shut down.(2)Genomic structure of PAF receptor.Human PAF receptor gene is present as a single copy in haploid on the chromosome 1q. The gene consists of three exons, separated each other > 20kb intron. Two5'-noncoding exons has distinct transcriptional start site, and promoters. These exons are alternatively spliced to a common splice acceptor to yield two different mRNAs. Transcript 1 is present in almost all tissues, and up-regulated by PAF or endotoxin through its NF-kB site. Transcript 2 is expressed in more tissue-specific way, and up-regulated by estrogen, and down-regulated by TGF-b. The physiological and pathological significance of these promoters are currently under investigation.